In 2012, this project provided state-of-the-art research technologies for NIAID's intramural infectious diseases, allergy, and immunology research programs. The new technologies are developed and validated and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the NIAID intramural program. The technologies supported include flow cytometry, confocal microscopy, DNA microarray, protein separation, mass spectrometry, peptide synthesis, protein sequencing, and macromolecular X-ray crystallography. Many of these technologies are used in high containment laboratories critical to the Institute's infectious diseases and biodefense research agenda. In addition to technology development, the RTB provides advanced training in all aspects of the technologies in the Branchs portfolio. Flow Cytometry The mission of the Flow Cytometry Section is to provide the DIR with application specific flow cytometric technologies, including instrumentation for high speed cell sorting and multi-color analysis, and to provide training, consultation, method development, and analysis for experiments involving flow cytometry. This mission is accomplished through the efforts of staff with extensive flow cytometry experience and with the use of eight cell sorters and eleven analyzers. Major goals are to provide access to state-of-the art technologies, to help design and run experiments, to facilitate data interpretation and to provide results that are of consistent high quality. Light Microscopy The mission of the Biological Imaging Section is to make available to DIR scientists advanced light microscopy imaging technologies and expertise in their use. This involves collaborating in both experimental design and instrument operation to best utilize the power of these cutting edge technologies, as well as to convey a basic understanding of the imaging process. These activities especially benefit those investigators whose specialty is not in microscopy or imaging. In practice, the Biological Imaging Section works collaboratively with laboratories within the DIR. Investigators are expected to participate in collection of their data, either as direct microscope operators or by selecting cells of interest, so that Biological Imaging Section staff can then collect the data. Often the collaboration begins much earlier, at the time of experimental design. The Biological Imaging Section advises on the most suitable instrument, appropriate labels, sample preparation, and best sampling regimens. Another responsibility of the Biological Imaging Section is to anticipate new directions in the imaging requirements of DIR and to insure that it is well-equipped and knowledgeable in those areas. The section also provides software and assistance with post collection image processing. Most frequently image analysis involves quantification, animation, 3D reconstruction, or segmentation. Protein Chemistry The Protein Chemistry Section develops project specific applications in the fields of peptide synthesis, N-terminal (Edman) sequencing, protein separation, assay development, and mass spectrometry. The RTB designs qualitative and quantitative physical-chemical and biochemical methods of detection and analysis that are customized to meet the specific research needs of DIR investigators. The Branch is also active in formulating and executing purification strategies for various types of molecules. This can also include customized small scale sample preparation and enrichment strategies for analysis by mass spectrometry. Another major activity of the RTB involves the development of more efficient and effective methods of sample preparation with a strong orientation towards mass spectroscopy to facilitate protein identification work. The Branch collaborates with DIR investigators to develop and validate protocols for protein separation and analysis for specific research programs of interest to the intramural research program. Genomic Technologies Genomic Technologies Section enables NIAID DIR investigators to utilize microarray and Next-Gen sequencing technology in their research programs. The section also provide statistical and bioinformatics data analysis support. The facilities maintain instrumentation and resources for Agilent and Illumina microarray platforms, Illumina Next-Gen nucleotide sequencing, commercial and customized bioinformatics software, and scientific computing. Recent collaborations have exploited our expertise in: Genome-wide assays of mRNA, micro-RNA, and DNA copy (CNV, CGH); Detection of DNA aberrations, DNA methylation, Epigenetics; Human disease biomarker discovery; Statistical analysis of microarray and high-throughput screening data; Bioinformatics, database mining, pathway and network analysis; Data management and analysis pipelines, reproducible data analysis protocols; Experiment design for microarray and other massively parallel assays. Structural Biology The mission of the Structural Biology Section is to provide specialized techniques and scientific expertise that enable DIR scientists to obtain structural information for macromolecules under study. This involves collaborating with DIR researchers to produce large quantities of highly purified proteins that are ready for crystallization, to prepare diffraction-quality crystals, and to determine X-ray crystal structures of proteins and other macromolecules that are central to their infectious diseases and immunology research programs. Investigators are expected to provide the macromolecular sample, in consultation with Structural Biology Section staff who will grow crystals and determine the X-ray structure. The Structural Biology Section can also express proteins and has experience in refolding proteins. The Structural Biology Section discusses the merits of planned structural determinations with DIR scientists, aiming to balance the efforts required to determine an X-ray structure with the insights predicted to derive from the structure. Depending on the collaborating group, investigators can be trained in protein production, crystallization, and X-ray crystallography.